Nanodiamond-dotma Complexes As Effective Non-viral Vectors For Gene Therapy

Alejandro Paredes, Nuseibah AL Qtaish Hasan, Myriam Sainz-Ramos, Tania Lopez-Mendez, Ilia Villate-Beitia, Idoia Gallego, Gustavo Puras, Jose Luis Pedraz

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Introduction: Gene therapies are rapidly becoming a critical component of the therapeutic armamentaria for a variety of inherited and acquired human diseases (1). Nevertheless, the development of safer and more efficient vectors for gene delivery remains an area of great potential for improvement. Nanodiamonds (ND) are a highly biocompatible material with unique physicochemical features that make them an attractive platform for the development of novel non-viral vectors (2). In this work, we describe for the first time the combination of a cationic lipid (DOTMA) and ND to gene delivery purposes.
Methods: ND-DOTMA vectors were obtained by an o/w emulsification method. The oil phase, formed by 1.25, 2.5, 5 or 7.5 mg of DOTMA diluted in 1 mL of dichloromethane, was added upon the aqueous phase, composed of 250 μL of NDs (10 mg/mL in water) and 3.75 mL of Tween® 20 0.27 %. Sonication was applied for 30 s (50 W) and subsequent solvent evaporation allowed obtaining formulations NTD10-13. Ternary complexes ND-DOTMA:DNA of several mass ratios were elaborated by mixing a pCMS-EGFP solution (0.5 mg/mL) with NTD formulations (Figure 1). Particle size, polydispersity index (PDI) and zeta potential (ζ) were determined in a Malvern Zetasizer®. Transfection experiments were performed in HEK-293 cell cultures following reported protocols (3), and the percentage of live transfected cells assayed by flow cytometry.
Results: NTD10-13 vectors presented particle sizes between 90 and 190 nm while PDI values were in the range of 0.177 and 0.231. ζ values were negative for the pure ND (–23.2 mV) and found to turn positive after emulsification with DOTMA (i.e. NTD13 44.4 mV). Complexation with DNA conducted to slight changes in particle sizes while ζ values remained positive. In cell culture experiments (Figure 2), NTD13 showed the most promising results with comparable percentages of transfection of the positive control LipofectamineTM 2000 (LIPO) (38.84 ± 3.36 and 43.26 ± 3.31 % respectively). Moreover, NTD13 showed a remarkable decrease in cell death when compared with LIPO (8.44 ± 3.80 and 20.60 ± 5.69 % respectively). NTD13 also showed a drop in cell death as compared to the controls without ND.
Conclusion: Ternary ND-DOTMA:DNA vectors were successfully prepared and characterized. A high transfection capacity was achieved and a remarkable decrease in cell toxicity was observed for ND formulations. Great potential for gene delivery is envisaged for these novel ND formulations and further in-vitro and in vivo studies are being carried out currently.
Acknowledgements: The financial, human and intellectual support from ICTS “NANBIOSIS” CIBER-BBN, SGIker, University of the Basque Country, and Basque Country Government is greatly appreciated.
References: (1) Dunbar CE, High K et al. Science. 2018, 359: 6372. (2) Leung, H. M. et al. ACS Sustain. Chem. Eng. 2018, 6: 9671–9681. (3) Ojeda E, Puras G, Agirre M, et al. Org Biomol Chem. 2015, 13(4): 1068-1081.
Original languageEnglish
Publication statusPublished - Jul 2020
EventCRS 2020 Virtual Annual Meeting - Las Vegas, USA, Las Vegas, United States
Duration: 29 Jun 202002 Jul 2020


ConferenceCRS 2020 Virtual Annual Meeting
Abbreviated titleCRS2020
Country/TerritoryUnited States
CityLas Vegas
Internet address


  • Nanodiamonds
  • gene therapy


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